Inflammasomes are innate immune sensing pathways designed to identify and clear infectious agents through production of pro-inflammatory mediators and pyroptotic cell death. Selective targeting of inflammasomes and their sensors, the NLRPs, is emerging as an important means of modulating the immune response in vaccinology, tumor therapy and treatment of autoimmunity. However, only two molecular mechanisms of NLRP activation have been described to date: NLRP1 proteolysis by Bacillus anthracis lethal factor and flagellin binding by the NAIPs/NLRC4 inflammasome. I have recently determined that NLRP1 also drives a host-protective inflammasome response to the protozoan parasite Toxoplasma gondii. In contrast to the previously described mechanism of NLRP1 activation by anthrax, however, NLRP1 is not proteolytically processed in response to Toxoplasma infection, leading to the hypothesis that NLRP1 has evolved to detect parasite infection via a novel, undescribed mechanism. This K22 award will provide the experimental resources, time and training to identify the critical components of the NLRP1 sensing pathway. Specifically, this award will allow me to develop 1) skills in the design and implementation of forward genetic screens, 2) tools and expertise in reverse genetic engineering of parasites, 3) biochemical tools to probe the essential components of NLRP1 for sensor activation. These studies address an understudied area in immunology: how eukaryotic pathogens are sensed by the innate immune system. Moreover, by exploiting the natural allelic variation in NLRP1 in parallel with a parasite genetics approach I will establish a unique set of tools to define the molecular underpinnings of NLRP1 activation that will be broadly important to our understanding of inflammasome biology. Specifically, the central goals of this project are to 1) identify the parasite components that trigger NLRP1 activation and 2) determine the critical residues in NLRP1 that mediate this response.